Pack fa kit

Fecal samples and cecum contents were collected 14 and 17 days after starting the pectin-supplemented diets, respectively. Each sample (20 mg) was suspended at a ratio of 1:50 (w/v) in MilliQ water, and disrupted using zirconia beads. After centrifugation at 10,000 × g for 10 min, SCFAs in the supernatant were measured using a YMC pack FA kit (YMC, Kyoto, Japan) and high-pressure liquid chromatography (HPLC) system (PU-2089 Plus, JASCO, Tokyo, Japan). The column was maintained at 50°C with a mobile phase consisting of acetonitrile-methanol-water (30:16:54 v/v/v, pH 4–5 adjusted by 0.1 M HCl) delivered at a flow rate of 0.5 mL/minute. Labeled SCFAs were detected at a wavelength of 400 nm using a UV/Visible HPLC detector (UV-2075 Plus, JASCO).

Cecal contents were obtained from 8-week-old mice that had been treated orally (or not) with an antibiotic cocktail (ampicillin, neomycin, and metronidazole each at 1 g/l, and vancomycin at 0.5 g/l) for 4 weeks. The samples were diluted in sterile water, mixed with 2-ethylbutyrate as an internal control, and then labeled with 2-nitrophenylhydrazine with the use of a YMC-Pack FA kit (YMC, Kyoto, Japan). SCFAs in the samples were analyzed by liquid chromatography (Prominence LC-20AD; Shimadzu, Kyoto, Japan).